Diseases of wildlife are increasingly threatening animal populations resulting in negative economic impacts, the loss of biodiversity, and risk to livestock and human health (Artois et al., 2009; Heard et al., 2013; Watsa, 2020). In some cases, disease has led to species extinctions (Skerratt et al., 2007; Heard et al., 2013); for example, the fungal parasite chytridiomycosis is a major contributor to global amphibian declines where over one third of amphibian species are now threatened or have already gone extinct (Cohen, 2003; Skerratt et al., 2007; Wake and Vredenburg, 2008). In other cases disease results in a major die off of already endangered populations, such as with Hemorrhagic septicemia, caused by a bacterial infection, in the endangered saiga antelope that resulted in the loss of half the remaining population in only a few weeks (Nicholls, 2015; Kock et al., 2018). Wildlife diseases have major economic impacts both from the spillover to domestic animals, such as Brucella from multiple wildlife species to domestic cattle, or the loss of game species; for example, chronic wasting disease in ungulates (Bengis et al., 2002; Wandeler et al., 2003; Bishop, 2004; Gomo et al., 2012; Zimmer et al., 2012; Kamath et al., 2016). Additionally, the majority of emerging infectious diseases in humans are a result of spillover from other animal species (Taylor et al., 2001; Belay et al., 2017). As a result we have seen a widespread increase in the need for identifying pathogens present in animal populations, defining disease dynamics among populations, determining spillover events, and the development of control strategies to mitigate disease spread (Artois et al., 2009; Ryser-Degiorgis, 2013; Watsa, 2020).

Wildlife pathogens surveillance is hampered by the difficulty of sampling a wide variety of species, many of which are elusive and challenging to capture. Overcoming this roadblock presents expensive and logistical challenges (Stallknecht, 2007; Watsa, 2020). Active disease surveillance, or the practice of direct sample collection for pathogen analysis, provides the greatest likelihood of detecting diseases in the population and limiting biases of data (Stallknecht, 2007; Artois et al., 2009). This approach is often costly and requires a significant amount of effort, thus limiting the number of species, number of animals, and the geographic area in which information can be collected (Stallknecht, 2007; Watsa, 2020). Opportunistic sample collection, termed “passive surveillance” can take advantage of sampling roadkill or other deceased animals, and presents a cost effective approach that can help to identify disease outbreaks (Hawkins et al., 2006; Lawson et al., 2015; Barron et al., 2018), this does however have its own caveats (Stallknecht, 2007; Santos et al., 2011). Carcasses can be quickly removed from the environment, via scavengers or other processes, undergo degradation when carcasses are not sampled in a timely manner or many carcasses are simply not detected and reported (Wobeser and Wobeser, 1992; Santos et al., 2011). In addition, samples often are biased towards animals that have been killed or are closer to human development (Ryser-Degiorgis, 2013). Hunter-collected sampling offers a promising approach to disease surveillance, as samples represent a large geographic representation of animals that can be sampled in a timely manner post-death (Brook et al., 2009; Lawson et al., 2015). Though this approach does still harbor some sample biases, for example favoring larger males (Belsare et al., 2020), disease data obtained from hunter samples can limit biases, logistical effort and costs.

Tooth samples are often required to be collected and submitted by hunters or directly collected by the permitting agency since morphological analyses can reveal age of the animal which is helpful for population management. Teeth can be relatively easily collected by trained technicians and sufficient amounts of DNA can be extracted from them for nucleic acid based diagnostic testing (Wandeler et al., 2003). The potential to recover pathogens from animal teeth remains largely unexplored, though in humans, pathogen data has been recovered from dental pulp of corpses (Barbieri et al., 2017). A benefit of using wildlife tooth samples is that potential to recover genomic information of pathogens, allowing characterization of pathogen strains and variants (Fitak et al., 2019). Genomic data from pathogens can inform their origin, geographic spread, cross-species transmissions, and host connectivity (Biek et al., 2006; Baele et al., 2018; Kraberger et al., 2020; Fountain-Jones et al., 2021). Increasingly, genomic data is used to determine spread in real time and identify environmental features associated with pathogen gene-flow (Holmes and Grenfell, 2009; Baele et al., 2017; Kozakiewicz et al., 2018), further highlighting the utility of obtaining pathogen genomic data routinely and with minimal cost from wildlife samples.

Pumas are a widespread large North American felid with varying management and population statuses that in the United States run a gamut, from endangered species status of the Florida panther and special protection in California, to game animal status in most western states, and complete lack of protection in Texas (“Non-game, Exotic, Endangered, Threatened and Protected Species” 2020, “Florida Panther – Florida Panther – U.S. Fish and Wildlife Service” n.d.). Pumas are capable of long distance movement (e.g., >1,000 Km over a lifetime (Hawley et al., 2016) and thus viral genomic signatures in specific areas may not translate to larger geographic regions. Infectious diseases have resulted in mortality of threatened puma populations, including viral spillover from domestic cats (Brown et al., 2008; Carver et al., 2016; Chiu et al., 2019; Kraberger et al., 2020). In particular, pumas are infected with several retroviruses that have been shown to reach high prevalence in populations, including a puma specific feline immunodeficiency virus (FIVpco) and feline foamy virus (FFV) (Franklin et al., 2007a, b; Lee et al., 2017; Kechejian et al., 2019). Assessment of viral infections of pumas has revealed cross-species transmission events, landscape features that influence viral structure, and disease-related die-offs in some populations (Lee et al., 2017; Chiu et al., 2019; Kraberger et al., 2020; Fountain-Jones et al., 2021). Genomic variation in FIVpco has been found to be structured, in part, by geographic location, and specific landscape features are associated with viral spread (Fountain-Jones et al., 2021) in contrast, genetic data of FFV has shown a lack of phylogenetic structure related to geography and instead show patterns of multiple spillover events from domestic cats to pumas (Kraberger et al., 2020). The difficulties in obtaining puma samples suitable for recovering diagnostic and genomic data from viruses has constrained these studies to a limited number of distinct geographic areas.

We analyzed DNA from teeth collected from 200 hunter-harvested pumas across Colorado to assess the capacity to use this tissue opportunistically for wildlife disease surveillance. We asked: (1) can viruses be detected in tooth samples of pumas? (2) is genomic material recovered of optimal quality for sequence analysis? and (3) how do viral sequences recovered from puma teeth inform geographical structure of viruses across the state compared to prior analyses conducted with active surveillance? This work illustrates that hunter-harvested samples utilized for disease surveillance provide pathogen data that is temporally linked, well-characterized with respect to individual animal demography, and spatially distinct.

Mitigation and control of wildlife disease is an increasingly important component of wildlife management. Hunter-harvested samples can be an important component of surveillance programs, and we demonstrate that hunter-collected teeth can not only determine infection status but permit the recovery of viral genetic information. Our results indicate that DNA extracted from teeth of wildlife is suitable for amplification of pathogen genomic sequences. This approach can be used to augment other sampling efforts to better inform the geographic structuring of viruses, and potentially other pathogens, at large spatial scales. The recovery of viral sequencing data from hunter-collected puma teeth demonstrate the utility of these samples to inform pathogen spread in harvested animals.

Few studies have evaluated presence of viruses from tooth samples, though both FIV and FFV infections of domestic cats are thought to be transmitted by bite wounds and thus can be detected in saliva (Miller et al., 2017; Cavalcante et al., 2018). Because viral infection may not be readily detected in the oral cavity, this methodology may not be suitable for evaluation of all pathogens (Bedarida et al., 2011; Siravenha et al., 2016). The ability to use tooth collected samples for diagnostics could be enhanced by targeting smaller regions of amplification. A comparison of tooth DNA and paired blood or lymphoid samples is required to truly compare sensitivity and specificity of both methods.

Feline immunodeficiency virus and feline foamy virus sequences isolated from tooth samples supported limited geographic structuring of retroviral infections of Colorado pumas across the state. FIVpco had more indication of geographic structuring than FFV, likely a result of the frequent cross-species transmission of FFV from domestic cats into pumas and low mutation rate (Kraberger et al., 2020). Phylogenies of FIVpco revealed individuals from the same geographic region tended to cluster together, consistent with previous results showing that in certain regions, spatial proximity was associated with spread of FIVpco (Fountain-Jones et al., 2021). The absence of clear geographic clusters indicates signals of phylogeographic clustering may dissipate with finer scale sampling efforts. These findings illustrate that Interstate 70, which bisects the state into northern and southern regions, and the continental divide, which bisects the state into eastern and western regions, were not impermeable to viral transmission. This is consistent with previous reports that high elevation ridgelines did not limit viral spread (Fountain-Jones et al., 2021) but in contrast to reduced geneflow noted across the continental divide, attributed to the fact that pumas avoid high ridgelines and alpine environments (Sweanor et al., 2000; Gustafson et al., 2019; Trumbo et al., 2019). Additionally, Interstate 70 did not have an apparent effect on viral structuring.

These results support the potential for genomes from rapidly evolving viral genomics to inform host connectivity more so than the genetics of the animals itself (Biek et al., 2006; Gagne et al., 2021). The results mirror studies evaluating interstate barriers to bobcat gene flow compared to FIVlru phylogeny, that illustrated Southern Californian interstates are more permeable to viral transmission than host gene flow (Lee et al., 2012; Ernest et al., 2014; Kozakiewicz et al., 2019, 2020). Because host genetic identities reflect dispersal related to reproductive events, it is less temporally sensitive than viral phylogeography, given the high evolutionary rates of viruses (Richardson et al., 2016; Gagne et al., 2021).

The addition of viral sequences from tooth samples demonstrated that potential geographic barriers (i.e., continental divide, major highways) did not appear to limit the spread of viruses in Colorado pumas. Our findings have important implications for puma population health should more virulent viruses, such as FeLV, arise in Colorado puma populations (Chiu et al., 2019). Further, this work illustrates a novel method for informing apex predator movement and may inform management of predator and prey populations. While additional validation is needed to determine the potential of tooth samples for diagnostic work that demonstrates pathogen prevalence, this work illustrates the benefits of incidentally-collected tooth samples as a tool for wildlife disease surveillance.

All sequence data has been uploaded to Genbank, NCBI (accession: MZ398368-MZ398467).

We handled all pumas in accordance with approved CPW Animal Care and Use Committee (ACUC) capture and handling protocols (ACUC file #08-2004, ACUC protocol #03-2007 ACUC 16-2008). Samples were provided to Colorado State University for diagnostic evaluation. Colorado State University and Colorado Parks and Wildlife (CPW) Institutional Animal Care and Use Committees reviewed and approved this work prior to initiation (CSU IACUC protocol 05-061A).

SK, SV, MA, and CA conceived the project. MA, KL, and CA conducted the field sampling and collections. SK, RG, RM, and KG performed the laboratory work. SK, RG, and DT analyzed the data. SK, RG, and SV wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.